The results provided in this thesis demonstrate that the -secretase inhibitors RO429097 or DAPT induce downregulation of the Notch signalling pathway in most hMSC cultures. This was demonstrated by a decrease in the expression of mRNA and protein coding for the Notch primary target HES1. This was further accompanied by upregulation of the RGC marker BRN3A/B, which is in agreement with previous reports that Notch inhibition causes RGC differentiation of these cells (Singhal et al., 2012, Becker et al., 2013). However, addition of TGFβ to hMSC MIO-M1 cultures undergoing RGC differentiation did not modify the expression of Notch target gene HES1 and RGC marker BRN3A/3B. Thus, as a future work, other hMSC cell lines undergoing RGC differentiation could be cultured with TGFβ1. Given the importance of TGFβ and Notch signalling during Müller glia derived neural differentiation in other species, it is possible that both these pathways may regulate hMSC RGC differentiation by interacting with other signalling pathways such as the Wnt pathway. Thus, the effect of TGFβ on hMSC RGC differentiation by interacting with other signalling pathways could be explored in future studies. Also, TGFβ signalling could be more important during early stages of hMSC RGC differentiation to regulate hMSC cell proliferation as reported in the zebrafish (Qin et al., 2009, Lenkowski and
differentiation cultured with TGFβ at various time points can be performed to understand the expression pattern in these cells.
The -secretase inhibitor RO4929097 downregulated the components of the canonical Wnt signalling including WNT2B, WISP-1, AXIN2 or DKK1 in hMSC cell lines MIO-M1 and 6387 respectively, agreeing with the results previously observed in the zebrafish (Ramachandran et al., 2012). However, no changes in WNT2B occurred in cell lines 6426 and 6391. Similarly, non-canonical Wnt ligand WNT5B in hMSC was only downregulated in MIO-M1 cells, but not in 6387. The variability observed in the effect of -secretase inhibitor on various hMSC lines may be attributed to the different condition of the donor tissues when used for isolating these cells. Therefore, although all hMSC lines retain various signalling pathways including the Wnt pathway, involved in stem cell differentiation and proliferation, but each cell line may possibly vary in the way these pathways can be modulated within these different hMSC lines. Taken together, the present results suggest the possible existence of a crosstalk between the components of Wnt and Notch signalling pathways in cultured hMSC. Moreover, the downregulation of the canonical Wnt signalling pathway may play an important role during hMSC RGC differentiation.
Further experiments using canonical Wnt inhibitors to hMSC cultured with RO4929097 will be required to test this hypothesis. Additionally, as a future study, it would be interesting to use this inhibitor and growth factor to retinal explants and analyse RGC or photoreceptor markers in retinal sections. Furthermore, these factors could also be injected in the damaged retina of newly born mice and the retinal cell markers analysed.
6.2
TGFβmodulates the expression of WNT signalling components in human Müller stem cells
Most retinal degenerative conditions that lead to blindness, including inflammatory, pro-angiogenic and dystrophic retinal diseases, have been associated with abnormal proliferation of Müller glia that does not lead to repair but to the formation of glial scarring (Bringmann et al., 2009). Many of these conditions are also accompanied by local increased production of pro-inflammatory factors such as TGFβ (Guo et al., 2014, Wang et al., 2013, Paine et al., 2012), which may potentially modify the neural progenicity of hMSC. During early development, TGFβ has been shown to synergize or antagonize with Wnt proteins, a family of highly conserved secreted signalling molecules that regulate cell-to-cell interactions (Nishita et al., 2000, Satterwhite and Neufeld, 2004, Tuli et al., 2003).
Activation of the canonical Wnt signaling by TGFβ has been shown to mediate fibrosis (Akhmetshina et al., 2012), and cooperation between TGFβ and Wnt signaling pathways are known to play a role in controlling developmental events such as the regulation of osteoblast differentiation of human mesenchymal stem cells (Zhou, 2011). Although interaction of these signalling pathways in fish and amphibians as well as small mammals during development and adult regeneration are documented, there is no knowledge about the interaction of these factors in the human Müller stem cells. On this basis, we investigated the role of TGFβ1 on the regulation of the Wnt signalling pathway in hMSC by examining the effect of this factor on the expression of the DKK1 and WNT2B and WNT5B ligands, previously shown to be expressed by mammalian Müller glial cells (Yi et al., 2007), as well as on the phosphorylation of β-catenin.
The results showed that TGFβ caused in vitro downregulation of the canonical Wnt signalling pathway in hMSC. This was demonstrated by a decrease in the expression of canonical Wnt signalling components WNT2B, DKK1 and active β-catenin cultured with this factor. Activation of the TGFβ and Wnt signalling pathways require the expression of specific receptors on the cell surface, and as previously shown, mammalian Müller glia express TGFβ and Wnt receptors and their ligands (Ikeda et al., 1998, Yafai et al., 2014, Liu et al., 2013, Yi et al., 2007), for which it is possible that activation of these pathways may trigger the neurogenic properties of human Müller glia as observed in other species. By contrast, non-canonical Wnt component WNT5B mRNA and protein expressions were increased by TGFs. The non-canonical Wnt signalling mediated by ligands such as WNT5B is known to inhibit canonical Wnt signalling by acting on β-catenin (Stoick-Cooper et al., 2007).
Thus, in hMSCs it may be possible that TGFβ signalling can regulate the canonical Wnt signalling pathway by increasing the expression levels of non-canonical Wnt signalling ligands such as WNT5B and subsequently inhibiting the canonical Wnt signalling pathway. Therefore, further investigations into the effect of non-canonical Wnt signalling ligands such as WNT5B on the downstream canonical Wnt signalling components in hMSC may give light into the involvement of this pathway.
Furthermore, to understand whether these findings are limited to in vitro conditions, the next step would be carrying out similar experiments under ex-vivo conditions or in animal models of retinal degeneration.
Furthermore, the effect of inhibition of TGFβ signalling on the Wnt signalling pathway was examined by using SB431542, an inhibitor of TGFβ type I receptor (ALK5) which selectively blocks the SMAD 2/3 dependent pathway (Inman et al., 2002) and
JNK inhibitor SP600125, which blocks SMAD independent pathway mediated through JNK transcription factor (Bennett et al., 2001). SB431542 antagonized the effect of this factor on the downregulation of WNT2B and the upregulation of WNT5B, whilst there was no observed alteration on the modulation of both ligands by TGFβ1 following the addition of the JNK inhibitor. These results indicate that modulation of the expression of WNT2B and WNT5B by TGFβ1 is caused by SMAD signalling activation. These findings suggest that TGFβ signalling may regulate the neurogenic ability of human Müller stem cells by modulating the components of the Wnt signalling pathway.